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The Role of the Endocannabinoid System in the Brain–Gut Axis

  • Keith A. Sharkey
    Correspondence
    Reprint requests Address requests for reprints to: Keith A. Sharkey, PhD, Department of Physiology and Pharmacology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada. fax: 403-283-2700.
    Affiliations
    Hotchkiss Brain Institute and Snyder Institute of Chronic Diseases, Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada
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  • John W. Wiley
    Affiliations
    Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan
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      The actions of cannabis are mediated by receptors that are part of an endogenous cannabinoid system. The endocannabinoid system (ECS) consists of the naturally occurring ligands N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), their biosynthetic and degradative enzymes, and the cannabinoid (CB) receptors CB1 and CB2. The ECS is a widely distributed transmitter system that controls gut functions peripherally and centrally. It is an important physiologic regulator of gastrointestinal motility. Polymorphisms in the gene encoding CB1 (CNR1) have been associated with some forms of irritable bowel syndrome. The ECS is involved in the control of nausea and vomiting and visceral sensation. The homeostatic role of the ECS also extends to the control of intestinal inflammation. We review the mechanisms by which the ECS links stress and visceral pain. CB1 in sensory ganglia controls visceral sensation, and transcription of CNR1 is modified through epigenetic processes under conditions of chronic stress. These processes might link stress with abdominal pain. The ECS is also involved centrally in the manifestation of stress, and endocannabinoid signaling reduces the activity of hypothalamic–pituitary–adrenal pathways via actions in specific brain regions, notably the prefrontal cortex, amygdala, and hypothalamus. Agents that modulate the ECS are in early stages of development for treatment of gastrointestinal diseases. Increasing our understanding of the ECS will greatly advance our knowledge of interactions between the brain and gut and could lead to new treatments for gastrointestinal disorders.

      Keywords

      Abbreviations used in this paper:

      2-AG (2-arachidonoylglycerol), ABHD (α/β-hydrolase domain–containing protein), CB (cannabinoid), CHS (cannabis hyperemesis syndrome), CNS (central nervous system), COX (cyclooxygenase), DAGL (diacylglycerol lipase), DNMT (DNA methyltransferase), ECS (endocannabinoid system), FAAH (fatty acid amide hydrolase), GI (gastrointestinal), HPA (hypothalamic–pituitary–adrenal), IBS (irritable bowel syndrome), MAGL (monoacylglycerol lipase), NAPE (N-arachidonoylphosphatidylethanolamine), PEA (palmitoylethanolamide), THC (Δ;9-tetrahydrocannabinol), TRPV (transient receptor potential vanilloid)
      Cannabis has been used for millennia to treat the symptoms of inflammatory and functional disorders of the gastrointestinal (GI) tract,
      • Grinspoon L.
      • Bakalar J.B.
      Marihuana, the Forbidden Medicine.
      including abdominal pain, cramps, diarrhea, nausea, and vomiting.
      • Aviello G.
      • Romano B.
      • Izzo A.A.
      Cannabinoids and gastrointestinal motility: animal and human studies.
      • Izzo A.A.
      • Sharkey K.A.
      Cannabinoids and the gut: new developments and emerging concepts.
      • Sharkey K.A.
      • Darmani N.A.
      • Parker L.A.
      Regulation of nausea and vomiting by cannabinoids and the endocannabinoid system.
      • Abalo R.
      • Vera G.
      • Lopez-Perez A.E.
      • et al.
      The gastrointestinal pharmacology of cannabinoids: focus on motility.
      • Alhouayek M.
      • Muccioli G.G.
      The endocannabinoid system in inflammatory bowel diseases: from pathophysiology to therapeutic opportunity.
      Currently, cannabis is used legally and illegally by patients seeking relief from a vast array of symptoms—many of GI origin. The availability and use of medical cannabis is increasing in the United States, Canada, and elsewhere, however, although cannabis is widely used for the treatment of nausea and abdominal pain, there is only limited evidence to support these uses.
      • Whiting P.F.
      • Wolff R.F.
      • Deshpande S.
      • et al.
      Cannabinoids for medical use: a systematic review and meta-analysis.
      About 25 years ago, researchers discovered the cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). Activation of these receptors accounts for the majority of the actions of the main psychoactive constituent of cannabis Δ9-tetrahydrocannabinol (THC).
      • Maccarrone M.
      • Bab I.
      • Biro T.
      • et al.
      Endocannabinoid signaling at the periphery: 50 years after THC.
      • Di Marzo V.
      • De Petrocellis L.
      Why do cannabinoid receptors have more than one endogenous ligand?.
      The CB1 and CB2, their ligands N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), and the enzymes that synthesize and degrade them are the major components of the endocannabinoid system (ECS).
      • Di Marzo V.
      • De Petrocellis L.
      Why do cannabinoid receptors have more than one endogenous ligand?.
      Our knowledge of the ECS has increased exponentially in recent years and we now appreciate its roles in the brain–gut axis and gut pathophysiology. The actions of the ECS appear to be largely homeostatic, contributing importantly to the regulation of motility and inflammation in the GI tract. However, activation of CB1 in the intestinal epithelium also contributes in what appears to be a maladaptive fashion to the development of metabolic disease and obesity.
      • Cani P.D.
      Crosstalk between the gut microbiota and the endocannabinoid system: impact on the gut barrier function and the adipose tissue.
      In the central nervous system (CNS), the ECS is involved in the pathophysiology of stress.
      • Hill M.N.
      • Patel S.
      • Campolongo P.
      • et al.
      Functional interactions between stress and the endocannabinoid system: from synaptic signaling to behavioral output.
      • Hill M.N.
      • Tasker J.G.
      Endocannabinoid signaling, glucocorticoid-mediated negative feedback, and regulation of the hypothalamic-pituitary-adrenal axis.
      We review the fundamental properties of the ECS and describe its role in peripheral mechanisms of visceral pain, focusing on the dorsal root ganglia, which contain the cell bodies of the sensory nerves that innervate the gut. In the ganglia that control visceral sensation, the gene encoding CB1 (CNR1) is regulated epigenetically under conditions of chronic stress—this finding might provide a mechanism that links stress with visceral pain. The ECS also controls nausea and vomiting, and yet, paradoxically, cannabis has been associated with a hyperemesis syndrome. New findings reveal ways in which the ECS can be manipulated to alter the regulation of gut motility and inflammation. Despite the challenges of targeting the ECS therapeutically, increasing our understanding of how the ECS regulates the brain–gut axis could provide new insights into mechanisms and treatments of GI diseases.

      Cannabinoids and the Endocannabinoid System

      Cannabis contains dozens of different cannabinoids, terpenes, pinenes, flavonoids, and other compounds—nearly 500 different chemicals.
      • Russo E.B.
      Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects.
      • Gertsch J.
      • Pertwee R.G.
      • Di Marzo V.
      Phytocannabinoids beyond the Cannabis plant—do they exist?.
      The best known for their biologic activities are THC and cannabidiol, but others, such as cannabichromene, Δ9-tetrahydrocannabivarin, cannabigerol, and cannabidivarin, are being increasingly studied for a variety of actions, including those in the GI tract.
      • De Petrocellis L.
      • Orlando P.
      • Moriello A.S.
      • et al.
      Cannabinoid actions at TRPV channels: effects on TRPV3 and TRPV4 and their potential relevance to gastrointestinal inflammation.
      • Izzo A.A.
      • Capasso R.
      • Aviello G.
      • et al.
      Inhibitory effect of cannabichromene, a major non-psychotropic cannabinoid extracted from Cannabis sativa, on inflammation-induced hypermotility in mice.
      The best-known receptors for these molecules include CB1 and CB2 (receptors for THC and most other cannabinoids), transient receptor potential vanilloid channels (which many cannabinoids interact with), G-protein–coupled receptor 55 (which binds some cannabinoids) and the 5-hydroxytryptamine receptor 1A (which is a receptor for cannabidiol). It is unlikely that all the receptors that mediate the actions of phytocannabinoids are known.
      • Hill A.J.
      • Williams C.M.
      • Whalley B.J.
      • et al.
      Phytocannabinoids as novel therapeutic agents in CNS disorders.
      • Di Marzo V.
      • Piscitelli F.
      The endocannabinoid system and its modulation by phytocannabinoids.
      Cannabis is active when inhaled, consumed, or administered by oromucosal spray or rectal suppository, but smoking is the most frequent method of delivery. Cannabinoids reach their target organs in different amounts or at different times, depending on their route of administration and metabolism.
      Endocannabinoids are synthesized from membrane lipids in response to specific signals—they are not stored in vesicles like most other neurotransmitters and hormones
      • Katona I.
      • Freund T.F.
      Multiple functions of endocannabinoid signaling in the brain.
      (Figure 1). Anandamide is normally synthesized from N-arachidonoylphosphatidylethanolamine (NAPE), which is formed by the transfer of arachidonic acid from the sn-1 position of a donor phospholipid to phosphatidylethanolamine by N-acyltransferase. Hydrolysis of NAPE by an N-acylphosphatidylethanolamine-hydrolyzing phospholipase D produces anandamide.
      • Di Marzo V.
      • Fontana A.
      • Cadas H.
      • et al.
      Formation and inactivation of endogenous cannabinoid anandamide in central neurons.
      There are, however, alternative pathways of anandamide synthesis.
      • Blankman J.L.
      • Cravatt B.F.
      Chemical probes of endocannabinoid metabolism.
      The principal enzyme for the degradation of anandamide is fatty acid amide hydrolase (FAAH).
      • Deutsch D.G.
      • Ueda N.
      • Yamamoto S.
      The fatty acid amide hydrolase (FAAH).
      FAAH and NAPE-hydrolyzing phospholipase D are found in the GI tract and CNS.
      • Di Marzo V.
      • Fontana A.
      • Cadas H.
      • et al.
      Formation and inactivation of endogenous cannabinoid anandamide in central neurons.
      • Marquez L.
      • Suarez J.
      • Iglesias M.
      • et al.
      Ulcerative colitis induces changes on the expression of the endocannabinoid system in the human colonic tissue.
      Figure thumbnail gr1
      Figure 1Distribution of enzymes involved in the biosynthesis and degradation of endocannabinoids in the nervous system. The role of 2-AG as a dominant retrograde synaptic transmitter and putative roles of anandamide (AEA) as an anterograde and intracellular transmitter. Anandamide is synthesized from NAPE by a specific phospholipase D and is degraded by FAAH. 2-AG is synthesized by DAGL-α and degraded by MAGL and ABHD-6 and ABHD-12. Anandamide and 2-AG can also be metabolized by COX2, which is shown in post-synaptic neurons but can also occur presynaptically. AA, arachidonic acid; AGs, 2-acylglycerols; DAGs, diacylglycerols; ER, endoplasmic reticulum; MAPK, mitogen-activated protein kinases; PIP2, phosphoinositide bisphosphate; PKA, protein kinase A; PPARs, peroxisome proliferator–activated receptors; TRPs, transient receptor potential channels; VGCCs, voltage-gated calcium channels. Dashed lines denote inactivation.
      Adapted from Di Marzo et al.
      • Di Marzo V.
      • Stella N.
      • Zimmer A.
      Endocannabinoid signalling and the deteriorating brain.
      Synthesis of 2-AG requires the activation of a phosphoinositol phospholipase C, which hydrolyzes inositol phospholipids at the sn-2 position to produce diacylglycerol.
      • Blankman J.L.
      • Cravatt B.F.
      Chemical probes of endocannabinoid metabolism.
      The hydrolysis of diacylglycerol via sn-1-selective diacylglycerol lipases (DAGL)-α (and potentially DAGL-β) then leads to the formation of 2-AG.
      • Blankman J.L.
      • Cravatt B.F.
      Chemical probes of endocannabinoid metabolism.
      There are also other pathways for synthesis of 2-AG. 2-AG is mostly degraded by monoacylglycerol lipase (MAGL), but in mouse brain, about 15% of the degradation is by the enzymes α/β-hydrolase domain-containing protein-6 (ABHD-6) and ABHD-12.
      • Blankman J.L.
      • Simon G.M.
      • Cravatt B.F.
      A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.
      Studies are needed to determine the relative activities of the enzymes that degrade 2-AG in the human brain and the GI tract. 2-AG can also be oxygenated by cy clooxygenase-2 (COX2) to form biologically active prostaglandin glyceryl esters, which regulate inflammation.
      • Hermanson D.J.
      • Gamble-George J.C.
      • Marnett L.J.
      • et al.
      Substrate-selective COX-2 inhibition as a novel strategy for therapeutic endocannabinoid augmentation.
      • Alhouayek M.
      • Muccioli G.G.
      COX-2-derived endocannabinoid metabolites as novel inflammatory mediators.
      Endocannabinoids signal via CB1 and CB2, which are present in the GI tract. These receptors have been detected on enteric nerves and the epithelium, and CB1 has been detected on some enteroendocrine cells. CB1 appears to be the receptor that is most active under physiologic conditions.
      • Izzo A.A.
      • Sharkey K.A.
      Cannabinoids and the gut: new developments and emerging concepts.
      • Trautmann S.M.
      • Sharkey K.A.
      The endocannabinoid system and its role in regulating the intrinsic neural circuitry of the gastrointestinal tract.
      Anandamide is a natural ligand of the transient receptor potential cation channel subfamily V member 1 (TRPV1).
      • Starowicz K.
      • Nigam S.
      • Di Marzo V.
      Biochemistry and pharmacology of endovanilloids.
      CB1 is one of the most abundant G-protein–coupled receptors in the brain, highlighting its importance in the control of central neurotransmission. It is also highly expressed in the enteric nervous system, on all classes of enteric neuron except inhibitory motor neurons.
      • Trautmann S.M.
      • Sharkey K.A.
      The endocannabinoid system and its role in regulating the intrinsic neural circuitry of the gastrointestinal tract.
      The widespread distribution of CB1 accounts for the broad range of activities of cannabinoid ligands and varied effects of cannabis. However, this widespread distribution also poses a considerable challenge to therapies that target CB1—ligands can have a large variety of effects on the central and peripheral nervous systems.
      CB2 is found on enteric neurons
      • Trautmann S.M.
      • Sharkey K.A.
      The endocannabinoid system and its role in regulating the intrinsic neural circuitry of the gastrointestinal tract.
      and also expressed by immune and epithelial cells in the GI tract.
      • Wright K.
      • Rooney N.
      • Feeney M.
      • et al.
      Differential expression of cannabinoid receptors in the human colon: cannabinoids promote epithelial wound healing.
      • Wright K.L.
      • Duncan M.
      • Sharkey K.A.
      Cannabinoid CB2 receptors in the gastrointestinal tract: a regulatory system in states of inflammation.
      Only a few studies have examined the role of CB2 in interactions between the brain and gut. One of the most interesting examined visceral pain. Oral administration of probiotic strains of Lactobacillus that reduced visceral sensitivity were found to up-regulate CB2 (and μ-opioid receptors) in the intestinal epithelium of rats. Blocking CB2 attenuates the reduction in visceral sensory thresholds caused by the alteration in the gut microbiome.
      • Rousseaux C.
      • Thuru X.
      • Gelot A.
      • et al.
      Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors.
      Little is known about the mechanisms of this fascinating effect.
      Similarly, chronic stress and antibiotic-induced dysbiosis up-regulated CNR2 messenger RNA (encodes CB2) in the GI tract.
      • Aguilera M.
      • Vergara P.
      • Martinez V.
      Stress and antibiotics alter luminal and wall-adhered microbiota and enhance the local expression of visceral sensory-related systems in mice.
      Although these are interesting observations, a clinical trial of the same Lactobacillus strains that reduced visceral sensitivity in rats found that in humans, these bacteria reduced expression of CB2.
      • Ringel-Kulka T.
      • Goldsmith J.R.
      • Carroll I.M.
      • et al.
      Lactobacillus acidophilus NCFM affects colonic mucosal opioid receptor expression in patients with functional abdominal pain—a randomised clinical study.
      However, the bacteria still appeared to reduce visceral sensation and up-regulate μ-opioid receptors. Studies are needed to determine whether this finding is specific to this probiotic only and whether CB2 regulates visceral sensation in humans.

      A Peripheral Mechanism Linking Chronic Stress to Pain

      Many patients with chronic visceral pain find that it is exacerbated by stress. There is increasing evidence that the ECS modulates chronic stress-associated increases in abdominal pain (visceral hyperalgesia)
      • Izzo A.A.
      • Sharkey K.A.
      Cannabinoids and the gut: new developments and emerging concepts.
      • Storr M.A.
      • Sharkey K.A.
      The endocannabinoid system and gut-brain signalling.
      (Figure 2).
      Figure thumbnail gr2
      Figure 2Effects of chronic stress on peripheral endocannabinoid pathways in visceral primary afferent neurons. Under conditions of chronic stress, levels of 2-AG and anandamide (AEA) increase, and endocannabinoid degradation enzymes COX-2 and FAAH are decreased in nociceptive dorsal root ganglia (DRG) neurons that innervate the colon and pelvis. Along with this, levels of CB1 are reduced and there is an increase in TRPV1 expression and phosphorylation in nociceptive primary afferent neurons. These effects are mediated by corticosteroids from the HPA pathway.
      CB1 localizes to the cell bodies of dorsal root ganglia neurons. Under baseline conditions, most of these are intermediate and larger-diameter neurons.
      • Hohmann A.G.
      • Herkenham M.
      Localization of central cannabinoid CB1 receptor messenger RNA in neuronal subpopulations of rat dorsal root ganglia: a double-label in situ hybridization study.
      • Bridges D.
      • Rice A.S.
      • Egertova M.
      • et al.
      Localisation of cannabinoid receptor 1 in rat dorsal root ganglion using in situ hybridisation and immunohistochemistry.
      There is, however, a population of small-diameter nociceptive neurons that express CB1 along with TRPV1 and contain peptides such as substance P and calcitonin gene-related peptide.
      • Bridges D.
      • Rice A.S.
      • Egertova M.
      • et al.
      Localisation of cannabinoid receptor 1 in rat dorsal root ganglion using in situ hybridisation and immunohistochemistry.
      • Ahluwalia J.
      • Urban L.
      • Capogna M.
      • et al.
      Cannabinoid 1 receptors are expressed in nociceptive primary sensory neurons.
      When there is inflammation at their peripheral terminals, the proportion of CB1-containing neurons increases and a much higher proportion co-localize with TRPV1.
      • Amaya F.
      • Shimosato G.
      • Kawasaki Y.
      • et al.
      Induction of CB1 cannabinoid receptor by inflammation in primary afferent neurons facilitates antihyperalgesic effect of peripheral CB1 agonist.
      Activation of CB1 inhibits pain perception (nociception), whereas activation of TRPV1 increases pain perception.
      • Malik Z.
      • Baik D.
      • Schey R.
      The role of cannabinoids in regulation of nausea and vomiting, and visceral pain.
      CB1 interacts with and inhibits TRPV1 directly and indirectly, via the cyclic AMP–protein kinase A pathway.
      • Turcotte D.
      • Le Dorze J.A.
      • Esfahani F.
      • et al.
      Examining the roles of cannabinoids in pain and other therapeutic indications: a review.
      In rats, chronic, intermittent water-avoidance stress was associated with reciprocal changes in levels of 2-AG and anandamide (increased) and endocannabinoid degradation enzymes COX2 and FAAH (decreased). In these rats, stress was also associated with a decrease in levels of CB1 and an increase in TRPV1 expression and phosphorylation in nociceptive lumbosacral primary afferent neurons innervating the colon and pelvis, but not in those innervating the lower extremities.
      • Hong S.
      • Fan J.
      • Kemmerer E.S.
      • et al.
      Reciprocal changes in vanilloid (TRPV1) and endocannabinoid (CB1) receptors contribute to visceral hyperalgesia in the water avoidance stressed rat.
      The reciprocal changes in CB1 and TRPV1 were reproduced by exposure of control sensory neurons with anandamide. In contrast, exposure of control dorsal root ganglia in vitro with the CB1 and CB2 agonist WIN 55,212-2 decreased the levels of TRPV1 and its phosphorylation. Treatment of stressed rats with WIN 55,212-2 or the TRPV1 antagonist capsazepine prevented visceral hyperalgesia.
      • Hong S.
      • Fan J.
      • Kemmerer E.S.
      • et al.
      Reciprocal changes in vanilloid (TRPV1) and endocannabinoid (CB1) receptors contribute to visceral hyperalgesia in the water avoidance stressed rat.
      Linkage of the CB1–TRPV1 pathway to hypothalamic–pituitary–adrenal (HPA) stress was confirmed in experiments demonstrating that administration of the glucocorticoid receptor antagonist RU-486 to stressed rats prevented the reciprocal changes in CB1 and TRPV1 expression and visceral hyperalgesia. Control rats given serial injections of corticosterone, which mimicked chronic stress, developed visceral hyperalgesia, had increases in anandamide content and expression of TRPV1, and decreased levels of CB1. These changes were prevented by RU-486.
      • Hong S.
      • Zheng G.
      • Wu X.
      • et al.
      Corticosterone mediates reciprocal changes in CB 1 and TRPV1 receptors in primary sensory neurons in the chronically stressed rat.
      Taken together these findings indicate that chronic stress induces visceral hyperalgesia that involves differential, region-specific changes in endovanilloid (an endogenous ligand for TRPV1) and endocannabinoid pathways in sensory neurons innervating the pelvic viscera. These observations could provide a link between chronic stress and visceral hyperalgesia and lead to new therapeutic approaches. They also raise the question, what sustains these chronic changes? There is evidence that these could be mediated by epigenetic changes in sensory neurons.

       Epigenetic Regulation of Chronic Stress-Induced Visceral Hyperalgesia

      Epigenetic modifications produce long-term effects on gene expression without altering the DNA sequence (Figure 3). Interest in epigenetic regulatory pathways has emerged rapidly because of their apparent significance in key physiological processes. Important epigenetic alterations include DNA methylation, catalyzed by DNA methyltransferases (DNMTs), such as DNMT1, which is responsible for the maintenance of methylation patterns, and DNMT3a and DNMT3b, which are responsible for de novo methylation patterns. Methylation typically silences genes, whereas histone acetylation, catalyzed by acetyltransferases, typically activates genes.
      • Bestor T.H.
      The DNA methyltransferases of mammals.
      • Miranda T.B.
      • Jones P.A.
      DNA methylation: the nuts and bolts of repression.
      • Karlic R.
      • Chung H.R.
      • Lasserre J.
      • et al.
      Histone modification levels are predictive for gene expression.
      DNA methylation and histone acetylation are considered to be stable yet reversible; the enzymes that control these processes can be induced or inhibited by biochemical and environmental factors.
      Figure thumbnail gr3
      Figure 3Effects of chronic stress on epigenetic regulation of the gene encoding CB1 (CNR1). Chromosomes are located in chromatin-bound territories in the nucleus. Euchromatin is characterized by DNase 1 hypersensitivity and specific combinations of histone marks that define active genomic regulatory elements, such as promoters H3K27ac + H3K4me3, and enhancers H3K27ac + H3K4me1. An enhancer can either increase or decrease transcription. Recent research demonstrates that, in brain, the DNA sequence CAC is a common site of methylation, in contrast to other tissues where CpG is most often methylated. Additionally, in brain, 5-hydroxymethylcytosine (5hmC), a reactive species, is methylated. In contrast, in the periphery, methylcytosine (hmC) is a common site for methylation. DNA methylation is catalyzed by DNMTs. Chronic stress is associated with increased levels of DNMT1-mediated methylation of CRN1, resulting in reduced expression. H3K4me3, histone H3 trimethyl Lys4; H3K27ac, histone H3 Lys27 acetylation; H3Kme1, histone H3 monomethyl Lys4.
      Adapted from Wiley et al.
      • Wiley J.W.
      • Higgins G.A.
      • Athey B.D.
      Stress and glucocorticoid receptor transcriptional programming in time and space: implications for the brain-gut axis.
      Chronic stress was associated with increased methylation of the Cnr1 gene promoter by DNMT1, resulting in reduced levels of CB1 in the sensory neurons that innervate the pelvic organs, including the colon, but not neurons that innervate the lower extremities
      • Hong S.
      • Zheng G.
      • Wiley J.W.
      Epigenetic regulation of genes that modulate chronic stress-induced visceral pain in the peripheral nervous system.
      (Figure 3). The glucocorticoid receptor (encoded by Nr3c1) is a transcription factor that also regulates the Cnr1 gene. Chronic stress was associated with increased DMNT1-mediated methylation of the Nr3c1 promoter and reduced expression of this gene in nociceptive primary afferent neurons.
      Also, chronic stress increases expression of the histone acetyltransferase EP300 and promotes acetylation of histones in the Trpv1 promoter, leading to increased levels of TRPV1 in these neurons.
      Region-specific knockdown of DNMT1 and EP300 gene expression in visceral primary afferent neurons innervating the pelvis reduced DNA methylation and histone acetylation, respectively, and prevented chronic stress-induced increases in visceral pain. These observations indicate that chronic stress promotes DNA methylation and down-regulation of anti-nociceptive Cnr1 and concurrent increase in histone acetylation of pro-nociceptive Trpv1 in a region- and cell-specific manner, resulting in visceral hyperalgesia.

      Endocannabinoids, Chronic Stress, and Pain

      The ECS is involved in the CNS response to stress and pain (Figure 4). Stress evokes changes in the levels of anandamide and 2-AG, reducing anandamide and increasing 2-AG.
      • Morena M.
      • Patel S.
      • Bains J.S.
      • et al.
      Neurobiological Interactions between stress and the endocannabinoid system.
      Chronic stress causes down-regulation or loss of CB1. These changes are widespread and occur throughout the brain.
      • Morena M.
      • Patel S.
      • Bains J.S.
      • et al.
      Neurobiological Interactions between stress and the endocannabinoid system.
      The functional consequences of this are the manifestation of the stress response, including activation of the HPA stress response and anxiety.
      • Morena M.
      • Patel S.
      • Bains J.S.
      • et al.
      Neurobiological Interactions between stress and the endocannabinoid system.
      Increased 2-AG signaling correlates with the termination and adaptation of the activity of the HPA axis and contributes to changes in pain perception. These effects presumably occur via remaining CB1 receptors, whose sensitivity may be increased.
      Figure thumbnail gr4
      Figure 4Effects of chronic stress on the endocannabinoid system in the brain. Chronic stress is characterized by a sustained reduction in levels of anandamide (AEA), associated with increases in the degradative enzyme FAAH. These effects are mediated by CRH via the CRH 1 receptor (CRHR1). Corticosterone (in rodents; corticosterol in humans; CORT) appear to mediate the up-regulation of CRH. The increased level of CORT are associated with increases in 2-AG. Chronic stress is associated with reduced levels of CB1 and the glucocorticoid receptor (GR) in the brain.
      Adapted from Morena et al.
      • Morena M.
      • Patel S.
      • Bains J.S.
      • et al.
      Neurobiological Interactions between stress and the endocannabinoid system.
      and Krugers et al.
      • Krugers H.J.
      • Hoogenraad C.C.
      • Groc L.
      Stress hormones and AMPA receptor trafficking in synaptic plasticity and memory.
      In support of the observation that endocannabinoids that act via the CB1 have an analgesic effect on pain signaling, inhibitors of endocannabinoid degradation reduce stress-related hyperalgesia.
      • Lomazzo E.
      • Bindila L.
      • Remmers F.
      • et al.
      Therapeutic potential of inhibitors of endocannabinoid degradation for the treatment of stress-related hyperalgesia in an animal model of chronic pain.
      Increasing anandamide signaling with URB597, a FAAH inhibitor, reduced chronic stress-induced anxiety and thermal hyperalgesia in mice. Endocannabinoid modulation by FAAH and MAGL also acts on the analgesic circuitry in the periaqueductal gray region of the brainstem to promote analgesia.
      • Lau B.K.
      • Drew G.M.
      • Mitchell V.A.
      • et al.
      Endocannabinoid modulation by FAAH and monoacylglycerol lipase within the analgesic circuitry of the periaqueductal grey.
      The periaqueductal gray is activated in response to noxious visceral stimulation
      • Wang Z.
      • Bradesi S.
      • Maarek J.M.
      • et al.
      Regional brain activation in conscious, nonrestrained rats in response to noxious visceral stimulation.
      and contributes to the descending inhibitory neural pathways that gate peripheral sensory input to the CNS. The individual or combined inhibition of FAAH and/or MAGL increased tonic disinhibition within the periaqueductal gray—an effect that is consistent with the promotion of analgesia. Supporting the concept that the combined inhibitor increases efficacy, the dual inhibitor of FAAH and MAGL JZL195 was more effective in reducing allodynia (central pain sensitization after painful, often repetitive, stimulation) than selective FAAH or MAGL inhibitors. The dual inhibitor also had a larger therapeutic window than cannabinoid receptor agonists in a mouse model of neuropathic pain
      • Adamson Barnes N.S.
      • Mitchell V.A.
      • Kazantzis N.P.
      • et al.
      Actions of the dual FAAH/MAGL inhibitor JZL195 in a murine neuropathic pain model.
      or in animal models of visceral pain.
      • Sakin Y.S.
      • Dogrul A.
      • Ilkaya F.
      • et al.
      The effect of FAAH, MAGL, and Dual FAAH/MAGL inhibition on inflammatory and colorectal distension-induced visceral pain models in Rodents.
      The endocannabinoid system appears to be sensitive to early-life stress, in a sex- and region-specific manner.
      • Marco E.M.
      • Echeverry-Alzate V.
      • Lopez-Moreno J.A.
      • et al.
      Consequences of early life stress on the expression of endocannabinoid-related genes in the rat brain.
      Early-life stress is an important factor in the development of irritable bowel syndrome (IBS) and is associated with epigenetic changes that lead to visceral hypersensitivity.
      • Moloney R.D.
      • Stilling R.M.
      • Dinan T.G.
      • et al.
      Early-life stress-induced visceral hypersensitivity and anxiety behavior is reversed by histone deacetylase inhibition.
      Male and female Wistar rats raised under standard conditions or exposed to maternal deprivation demonstrated changes in the ECS. Specifically, expression of genes in the cannabinoid system (Cnr1, Cnr2a, Cnr2b, and GPR55, which encode cannabinoid receptors and GPR55) were measured in adolescent rats in different regions of brain (frontal cortex, ventral and dorsal striatum, dorsal hippocampus, and amygdala). Maternal deprivation increased the expression of all the endocannabinoid genes surveyed in the frontal cortex in male rats, whereas in female rats, increased expression was noted only in the hippocampus. These observations have potentially important implications for the differential sex-specific effects of stress on pain perception and the mechanisms by which early life affects these responses.
      Diurnal changes are a feature of visceral sensation and correlate with changes in the HPA axis,
      • Gschossmann J.M.
      • Buenger L.
      • Adam B.
      • et al.
      Diurnal variation of abdominal motor responses to colorectal distension and plasma cortisol levels in rats.
      which is dysregulated in patients with IBS.
      • Chang L.
      • Sundaresh S.
      • Elliott J.
      • et al.
      Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in irritable bowel syndrome.
      The ECS regulates basal and circadian HPA axis activation. For example, the hypothalamic content of anandamide is highest at 7:00 am and 11:00 am and low between 3:00 pm and 3:00 am.
      • Liedhegner E.S.
      • Sasman A.
      • Hillard C.J.
      Brain region-specific changes in N-acylethanolamine contents with time of day.
      In vivo studies demonstrate that low doses of CB1 agonists other than THC reduced basal and stress-induced HPA pathway responses in rodents.
      • Patel S.
      • Roelke C.T.
      • Rademacher D.J.
      • et al.
      Endocannabinoid signaling negatively modulates stress-induced activation of the hypothalamic-pituitary-adrenal axis.
      • Saber-Tehrani A.
      • Naderi N.
      • Hosseini Najarkolaei A.
      • et al.
      Cannabinoids and their interactions with diazepam on modulation of serum corticosterone concentration in male mice.
      In persons who were either naïve to cannabis or infrequent users, acute consumption of cannabis
      • Cone E.J.
      • Johnson R.E.
      • Moore J.D.
      • et al.
      Acute effects of smoking marijuana on hormones, subjective effects and performance in male human subjects.
      or THC
      • Kleinloog D.
      • Liem-Moolenaar M.
      • Jacobs G.
      • et al.
      Does olanzapine inhibit the psychomimetic effects of Delta(9)-tetrahydrocannabinol?.
      • Klumpers L.E.
      • Beumer T.L.
      • van Hasselt J.G.
      • et al.
      Novel Delta(9) -tetrahydrocannabinol formulation Namisol(R) has beneficial pharmacokinetics and promising pharmacodynamic effects.
      increased secretion of cortisol. The stimulatory effect of THC administration on cortisol levels was lower in chronic cannabis users, supporting the development of tolerance.
      • D'Souza D.C.
      • Ranganathan M.
      • Braley G.
      • et al.
      Blunted psychotomimetic and amnestic effects of delta-9-tetrahydrocannabinol in frequent users of cannabis.
      • Ranganathan M.
      • Braley G.
      • Pittman B.
      • et al.
      The effects of cannabinoids on serum cortisol and prolactin in humans.
      Some studies
      • King G.R.
      • Ernst T.
      • Deng W.
      • et al.
      Altered brain activation during visuomotor integration in chronic active cannabis users: relationship to cortisol levels.
      • Somaini L.
      • Manfredini M.
      • Amore M.
      • et al.
      Psychobiological responses to unpleasant emotions in cannabis users.
      found that chronic cannabis users had higher basal levels of cortisol than infrequent users. Stress-induced activation of the HPA stress response was reported to be reduced in adult and adolescent chronic users of cannabis.
      • Somaini L.
      • Manfredini M.
      • Amore M.
      • et al.
      Psychobiological responses to unpleasant emotions in cannabis users.
      • van Leeuwen A.P.
      • Creemers H.E.
      • Greaves-Lord K.
      • et al.
      Hypothalamic-pituitary-adrenal axis reactivity to social stress and adolescent cannabis use: the TRAILS study.
      Taken together, these observations indicate that chronic cannabis use might dysregulate basal, circadian, and stress-regulated HPA stress responses in a complex manner.
      The ECS suppresses the activity of the HPA pathway via distinct actions within the prefrontal cortex, amygdala, hypothalamus, and hippocampus.
      • Hill M.N.
      • Tasker J.G.
      Endocannabinoid signaling, glucocorticoid-mediated negative feedback, and regulation of the hypothalamic-pituitary-adrenal axis.
      The region-specific action of the ECS in the CNS could account for the observed effects of cannabinoids on anxiety, appetite, nausea, and pain.

      Endocannabinoids in the Control of Nausea and Vomiting

      Cannabis is an effective anti-emetic and is used in the clinic, although it is not a first-line therapeutic agent.
      • Sharkey K.A.
      • Darmani N.A.
      • Parker L.A.
      Regulation of nausea and vomiting by cannabinoids and the endocannabinoid system.
      However, unlike most commonly used anti-emetics, cannabis also prevents or limits the sensation of nausea. The brainstem circuitry of vomiting is well established.
      • Sharkey K.A.
      • Darmani N.A.
      • Parker L.A.
      Regulation of nausea and vomiting by cannabinoids and the endocannabinoid system.
      The ECS is involved in the control of the emetic reflex, as shown by the fact that antagonists of CB1 cause vomiting in humans and exacerbate it animals. Interestingly, not only do CB1 agonists block emesis, but so do exogenous and endogenous agonists of CB2.
      • Van Sickle M.D.
      • Duncan M.
      • Kingsley P.J.
      • et al.
      Identification and functional characterization of brainstem cannabinoid CB2 receptors.
      Because CB2 activation is not psychotropic, this observation potentially opens up new avenues for therapeutic interventions.
      Unlike vomiting, the brain circuitry responsible for evoking nausea is incompletely understood, but is being elucidated through the use of functional magnetic resonance imaging in humans and through the use of animal models.
      • Sharkey K.A.
      • Darmani N.A.
      • Parker L.A.
      Regulation of nausea and vomiting by cannabinoids and the endocannabinoid system.
      • Napadow V.
      • Sheehan J.D.
      • Kim J.
      • et al.
      The brain circuitry underlying the temporal evolution of nausea in humans.
      • Sclocco R.
      • Kim J.
      • Garcia R.G.
      • et al.
      Brain circuitry supporting multi-organ autonomic outflow in response to nausea.
      These studies have associated nausea with activation of many complex neural circuits involving brainstem, limbic, interoceptive, somatosensory, and cognitive neural networks.
      • Napadow V.
      • Sheehan J.D.
      • Kim J.
      • et al.
      The brain circuitry underlying the temporal evolution of nausea in humans.
      • Sclocco R.
      • Kim J.
      • Garcia R.G.
      • et al.
      Brain circuitry supporting multi-organ autonomic outflow in response to nausea.
      In a rat model of nausea, when levels of endocannabinoid were increased systemically by administration of an FAAH inhibitor, nausea was reduced. However, this effect is not mediated by CB1
      • Sticht M.A.
      • Limebeer C.L.
      • Rafla B.R.
      • et al.
      Endocannabinoid regulation of nausea is mediated by 2-arachidonoylglycerol (2-AG) in the rat visceral insular cortex.
      • Parker L.A.
      • Rock E.M.
      • Sticht M.A.
      • et al.
      Cannabinoids suppress acute and anticipatory nausea in preclinical rat models of conditioned gaping.
      because it was not blocked by an antagonist. In contrast, inhibition of MAGL, which increases levels of 2-AG, is mediated by CB1. Studies of humans and animals have found the insular cortex to be involved in the perception of nausea (human studies) and physiologic responses to nausea (animal studies).
      • Napadow V.
      • Sheehan J.D.
      • Kim J.
      • et al.
      The brain circuitry underlying the temporal evolution of nausea in humans.
      • Sclocco R.
      • Kim J.
      • Garcia R.G.
      • et al.
      Brain circuitry supporting multi-organ autonomic outflow in response to nausea.
      • Parker L.A.
      Conditioned flavor avoidance and conditioned gaping: rat models of conditioned nausea.
      Activation of the ECS in the insular cortex can regulate the expression of nausea in animals; interestingly, it appears that 2-AG, rather than anandamide, mediates this regulatory control system
      • Sticht M.A.
      • Limebeer C.L.
      • Rafla B.R.
      • et al.
      Endocannabinoid regulation of nausea is mediated by 2-arachidonoylglycerol (2-AG) in the rat visceral insular cortex.
      • Sticht M.A.
      • Limebeer C.L.
      • Rafla B.R.
      • et al.
      Intra-visceral insular cortex 2-arachidonoylglycerol, but not N-arachidonoylethanolamide, suppresses acute nausea-induced conditioned gaping in rats.
      (Figure 5). MAGL inhibitors, but not FAAH inhibitors, when administered into the visceral insular cortex, block nausea in animals.
      • Sticht M.A.
      • Limebeer C.L.
      • Rafla B.R.
      • et al.
      Endocannabinoid regulation of nausea is mediated by 2-arachidonoylglycerol (2-AG) in the rat visceral insular cortex.
      • Sticht M.A.
      • Limebeer C.L.
      • Rafla B.R.
      • et al.
      Intra-visceral insular cortex 2-arachidonoylglycerol, but not N-arachidonoylethanolamide, suppresses acute nausea-induced conditioned gaping in rats.
      Further studies are needed to elucidate how the selectivity occurs in this system, when both endocannabinoid ligands are agonists of CB1, but the process could involve locations of the receptors and/or metabolism of endocannabinoids in this brain region. It will also be important to determine whether agents that alter the ECS system, beyond cannabis, can be developed for treatment of patients with nausea.
      Figure thumbnail gr5
      Figure 5Role of endocannabinoids in control of nausea and vomiting. In the brainstem regions involved in the control of vomiting (area postrema and nucleus of the solitary tract), activation of CB1 and possibly CB2 attenuates the emetic reflex by reducing the release of excitatory transmitters. In the forebrain, release of 2-AG in the visceral insular cortex inhibits, in a retrograde manner, release of neurotransmitters including serotonin (5-HT), which acts at 5-HT3 receptors to produce nausea. Note that to date this model has been studied in the rat and that in humans the CB1 agonist Δ9-tetrahydrocannabinol reduces nausea. 5-HT3 receptor antagonists are excellent antiemetics but are less effective at reducing nausea.
      Adapted from Sticht et al.
      • Sticht M.A.
      • Rock E.M.
      • Limebeer C.L.
      • et al.
      Endocannabinoid mechanisms influencing nausea.
      The cannabinoid hyperemesis syndrome (CHS) was first described in 2004.
      • Allen J.H.
      • de Moore G.M.
      • Heddle R.
      • et al.
      Cannabinoid hyperemesis: cyclical hyperemesis in association with chronic cannabis abuse.
      CHS is a syndrome of cyclic episodes of nausea, vomiting, and abdominal pain associated with chronic cannabis usage. CHS is frequently confused with cyclic vomiting syndrome, which has similar features.
      • Lee L.Y.
      • Abbott L.
      • Moodie S.
      • et al.
      Cyclic vomiting syndrome in 28 patients: demographics, features and outcomes.
      An interesting and poorly understood feature of the syndrome is that patients report reduced symptoms after hot baths or showers.
      • Allen J.H.
      • de Moore G.M.
      • Heddle R.
      • et al.
      Cannabinoid hyperemesis: cyclical hyperemesis in association with chronic cannabis abuse.
      • Chang Y.H.
      • Windish D.M.
      Cannabinoid hyperemesis relieved by compulsive bathing.
      • Soriano-Co M.
      • Batke M.
      • Cappell M.S.
      The cannabis hyperemesis syndrome characterized by persistent nausea and vomiting, abdominal pain, and compulsive bathing associated with chronic marijuana use: a report of eight cases in the United States.
      Typically, standard anti-emetics are ineffective in treating CHS.
      • Wallace E.A.
      • Andrews S.E.
      • Garmany C.L.
      • et al.
      Cannabinoid hyperemesis syndrome: literature review and proposed diagnosis and treatment algorithm.
      Little is known about the mechanism(s) of this syndrome—they might involve toxic metabolite(s) from the cannabis plant or progressive high exposure to the ligand can lead to down-regulation of cannabinoid receptors and loss of the endocannabinoid anti-emetic pathway.
      • Sharkey K.A.
      • Darmani N.A.
      • Parker L.A.
      Regulation of nausea and vomiting by cannabinoids and the endocannabinoid system.
      CHS has not been reported in patients taking only a pure THC product, such as dronabinol, on a scheduled regimen. Genetic factors might also affect susceptibility to CHS. Patients who experience hyperemesis from chronic cannabis use might have a genetic variation in their hepatic drug-transforming enzymes that results in excessive levels of cannabis metabolites that promote emesis.
      • Darmani N.A.
      Cannabinoid-induced hyperemesis: a conundrum—from clinical recognition to basic science mechanisms.
      The cutaneous (cutaneous steal) and splanchnic (vasodilation) circulations have been proposed as contributing factors to abdominal pain and nausea.
      • Bolognesi M.
      • Di Pascoli M.
      • Verardo A.
      • et al.
      Splanchnic vasodilation and hyperdynamic circulatory syndrome in cirrhosis.
      Currently, the only effective treatment of CHS is a drug “holiday”—cessation of cannabis use to allow clearance of putative toxic metabolites and/or normalization of CB receptor levels and function.

      Cannabinoids and Control of Gastrointestinal Motility

      Cannabis reduces cramping and slows GI motility. These actions prompted detailed studies of the role of the ECS in the control of motility and specifically in IBS (Figure 6). Variants of the CNR1 and FAAH genes have been observed in patients with diarrhea-predominant and alternating forms of IBS.
      • Camilleri M.
      • Kolar G.J.
      • Vazquez-Roque M.I.
      • et al.
      Cannabinoid receptor 1 gene and irritable bowel syndrome: phenotype and quantitative traits.
      • Park J.M.
      • Choi M.G.
      • Cho Y.K.
      • et al.
      Cannabinoid receptor 1 gene polymorphism and irritable bowel syndrome in the Korean population: a hypothesis-generating study.
      • Camilleri M.
      • Carlson P.
      • McKinzie S.
      • et al.
      Genetic variation in endocannabinoid metabolism, gastrointestinal motility, and sensation.
      Levels of FAAH messenger RNA are reduced in intestinal tissues of patients with constipation-predominant IBS.
      • Fichna J.
      • Wood J.T.
      • Papanastasiou M.
      • et al.
      Endocannabinoid and cannabinoid-like fatty acid amide levels correlate with pain-related symptoms in patients with IBS-D and IBS-C: a pilot study.
      These genetic polymorphisms and alterations in gene expression are associated with alterations in GI motility and sensation, supporting the pathophysiologic significance of alterations in the ECS in the gut.
      Figure thumbnail gr6
      Figure 6Effects of endocannabinoids in the GI tract are mediated by CB1 and CB2. Cannabinoid receptors are widely distributed in the GI tract. CB1 is expressed by all classes of cholinergic enteric neurons in the myenteric and submucosal plexuses (primary afferent neurons [blue], interneurons [purple], secretomotor [yellow], and excitatory motor neurons [green], but not on inhibitory motor neurons [red]). CB1 is also found on some enteroendocrine cells and in the epithelium. Extrinsic vagal and spinal primary afferent neurons express CB1, which is regulated by feeding (vagal) and stress (spinal), respectively. CB2 is expressed by enteric neurons and immune cells in the GI tract. Under conditions of inflammation and injury, CB2 is up-regulated in the epithelium and its function on enteric neurons increases. The classes of enteric neurons that express CB2 have not been determined. The effects of activating cannabinoid receptors in the GI tract include a reduction in motility, reduced inflammation, and reduced immune activation.
      Adapted from Lomax et al.
      • Lomax A.E.
      • Linden D.R.
      • Mawe G.M.
      • et al.
      Effects of gastrointestinal inflammation on enteroendocrine cells and enteric neural reflex circuits.
      In mice, disruption of Cnr1 increases motility. Consistent with this, patients given CB1 antagonists report diarrhea.
      • Izzo A.A.
      • Sharkey K.A.
      Cannabinoids and the gut: new developments and emerging concepts.
      Remarkably, disruption of Cnr1 specifically in the vagal system (afferent and efferent neurons) also increased GI motility,
      • Vianna C.R.
      • Donato Jr., J.
      • Rossi J.
      • et al.
      Cannabinoid receptor 1 in the vagus nerve is dispensable for body weight homeostasis but required for normal gastrointestinal motility.
      but it is not clear whether this was due only to changes in gastric emptying. Nonetheless, the ECS in the gut appears to be a potential therapeutic target for IBS and other functional bowel disorders.
      Researchers have studied whether peripherally restricted cannabinoids or locally produced endocannabinoids alters gut motility. Potent agonists of CB1 without central actions have been examined in rats and mice with increased GI motility caused by stress, as well as in control animals.
      • Keenan C.M.
      • Storr M.A.
      • Thakur G.A.
      • et al.
      AM841, a covalent cannabinoid ligand, powerfully slows gastrointestinal motility in normal and stressed mice in a peripherally restricted manner.
      • Abalo R.
      • Chen C.
      • Vera G.
      • et al.
      In vitro and non-invasive in vivo effects of the cannabinoid-1 receptor agonist AM841 on gastrointestinal motor function in the rat.
      • Cluny N.L.
      • Keenan C.M.
      • Duncan M.
      • et al.
      Naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (SAB378), a peripherally restricted cannabinoid CB1/CB2 receptor agonist, inhibits gastrointestinal motility but has no effect on experimental colitis in mice.
      Interestingly, agonists were more potent in stressed than control mice, and extremely low doses (which had no effects in control mice) were found to normalize accelerated small- and large-intestinal motility.
      • Keenan C.M.
      • Storr M.A.
      • Thakur G.A.
      • et al.
      AM841, a covalent cannabinoid ligand, powerfully slows gastrointestinal motility in normal and stressed mice in a peripherally restricted manner.
      There is no clear explanation for these observations, but possibilities include either an increase in numbers or affinity of CB1, or the coupling of the second messenger systems to the effector responses.
      Naturally released endocannabinoids might potentially be used to control GI motility. Drugs that increase or reduce the local levels of endocannabinoids, which would have activities in only the specific areas where they are produced, could theoretically have limited off-target actions. Proof of concept for this idea comes from studies using DAGL and FAAH inhibitors. Inhibitors of DAGL have been examined in models of constipation; they effectively normalized slowed transit in opioid-induced constipation and in congenital slow-transit constipation.
      • Bashashati M.
      • Nasser Y.
      • Keenan C.M.
      • et al.
      Inhibiting endocannabinoid biosynthesis: a novel approach to the treatment of constipation.
      Conversely, inhibiting the degradation of endocannabinoids by blocking FAAH has antidiarrheal and anti-nociceptive effects in models of accelerated motility and in mice with oil of mustard-induced colitis, respectively.
      • Fichna J.
      • Salaga M.
      • Stuart J.
      • et al.
      Selective inhibition of FAAH produces antidiarrheal and antinociceptive effect mediated by endocannabinoids and cannabinoid-like fatty acid amides.
      • Bashashati M.
      • Storr M.A.
      • Nikas S.P.
      • et al.
      Inhibiting fatty acid amide hydrolase normalizes endotoxin-induced enhanced gastrointestinal motility in mice.
      In these studies, increasing levels of endocannabinoids in the gut was not associated with cannabinoid-like side effects, such as reduced ambulatory locomotion. However, it should be noted that these examples are both acute studies—chronic administration can have negative effects because of the physiologic role of the ECS. For example, inhibiting FAAH can promote cardiovascular injury because of the actions of the ECS in the heart, including atherosclerotic plaque formation and myocardial tissue injury.
      • Mukhopadhyay P.
      • Horvath B.
      • Rajesh M.
      • et al.
      Fatty acid amide hydrolase is a key regulator of endocannabinoid-induced myocardial tissue injury.
      • Hoyer F.F.
      • Khoury M.
      • Slomka H.
      • et al.
      Inhibition of endocannabinoid-degrading enzyme fatty acid amide hydrolase increases atherosclerotic plaque vulnerability in mice.
      • Lenglet S.
      • Thomas A.
      • Soehnlein O.
      • et al.
      Fatty acid amide hydrolase deficiency enhances intraplaque neutrophil recruitment in atherosclerotic mice.
      Nonetheless, peripherally restricted cannabinoids and modulators of endocannabinoid synthesis and/or degradation might be used for short-term treatment of symptoms of IBS and other functional bowel disorders.

      Regulation of Intestinal Inflammation by Cannabinoids and Fatty Acid Ethanolamides

      The homeostatic role of the ECS also extends to the control of intestinal inflammation (Figure 6). Although exogenous cannabinoids can reduce colitis, one therapeutic strategy has been to increase levels of endocannabinoids by inhibiting their degradation. Inhibiting FAAH, which increases the levels of anandamide and other fatty acid ethanolamides, blocks the development of colitis in mouse models of inflammatory bowel disease.
      • Massa F.
      • Marsicano G.
      • Hermann H.
      • et al.
      The endogenous cannabinoid system protects against colonic inflammation.
      • Storr M.A.
      • Keenan C.M.
      • Emmerdinger D.
      • et al.
      Targeting endocannabinoid degradation protects against experimental colitis in mice: involvement of CB1 and CB2 receptors.
      • Salaga M.
      • Mokrowiecka A.
      • Zakrzewski P.K.
      • et al.
      Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition of fatty acid amide hydrolase (FAAH).
      Evidence in support of the homeostatic role of this system comes from studies of FAAH knockout mice, which develop a significantly milder colitis than control mice.
      • Massa F.
      • Marsicano G.
      • Hermann H.
      • et al.
      The endogenous cannabinoid system protects against colonic inflammation.
      The anti-inflammatory effects of FAAH inhibition are mediated by CB1 and CB2. Inhibitors of MAGL also block the development of colitis in mice,
      • Alhouayek M.
      • Lambert D.M.
      • Delzenne N.M.
      • et al.
      Increasing endogenous 2-arachidonoylglycerol levels counteracts colitis and related systemic inflammation.
      but more extensive investigations of the homeostatic role of MAGL are required.
      These studies raised the question of whether colitis develops, in part, through the loss of this endogenous anti-inflammatory system. Although polymorphisms in FAAH have not been directly associated with risk for inflammatory bowel disease,
      • Storr M.
      • Emmerdinger D.
      • Diegelmann J.
      • et al.
      The role of fatty acid hydrolase gene variants in inflammatory bowel disease.
      patients with Crohn’s disease homozygous for the mutation in FAAH encoding the amino acid change Pro129Thr were more likely to develop severe disease associated with fistulae and extra-intestinal manifestations. Patients with ulcerative colitis who are homozygous for this FAAH variant had earlier onset of disease than patients without this variant.
      • Storr M.
      • Emmerdinger D.
      • Diegelmann J.
      • et al.
      The role of fatty acid hydrolase gene variants in inflammatory bowel disease.
      Patients with acute ulcerative colitis have an increase in FAAH-reactive immune cells in the lamina propria,
      • Marquez L.
      • Suarez J.
      • Iglesias M.
      • et al.
      Ulcerative colitis induces changes on the expression of the endocannabinoid system in the human colonic tissue.
      but FAAH expression has been reported to be increased or reduced in different studies.
      • Salaga M.
      • Mokrowiecka A.
      • Zakrzewski P.K.
      • et al.
      Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition of fatty acid amide hydrolase (FAAH).
      • Suarez J.
      • Romero-Zerbo Y.
      • Marquez L.
      • et al.
      Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids.
      Similarly, in patients with colitis, levels of anandamide have also been reported to be either increased or reduced.
      • D'Argenio G.
      • Valenti M.
      • Scaglione G.
      • et al.
      Up-regulation of anandamide levels as an endogenous mechanism and a pharmacological strategy to limit colon inflammation.
      • Di Sabatino A.
      • Battista N.
      • Biancheri P.
      • et al.
      The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease.
      Further studies are needed to reconcile these disparate findings.
      Recently, a dual FAAH and COX inhibitor was evaluated in mice with colitis.
      • Sasso O.
      • Migliore M.
      • Habrant D.
      • et al.
      Multitarget fatty acid amide hydrolase/cyclooxygenase blockade suppresses intestinal inflammation and protects against nonsteroidal anti-inflammatory drug-dependent gastrointestinal damage.
      This interesting compound reduced the severity of colitis by increasing levels of anandamide, which acted on CB1, and also increased levels of the fatty acid ethanolamides palmitoylethanolamide (PEA) and oleoylethanolamide, which act via peroxisome proliferator-activated receptor–α. Levels of PEA are increased in intestinal tissues of patients with ulcerative colitis.
      • Darmani N.A.
      • Izzo A.A.
      • Degenhardt B.
      • et al.
      Involvement of the cannabimimetic compound, N-palmitoyl-ethanolamine, in inflammatory and neuropathic conditions: review of the available pre-clinical data, and first human studies.
      PEA is a powerful anti-inflammatory agent that reduces features of colitis in mice and secretion of inflammatory cytokines.
      • Borrelli F.
      • Romano B.
      • Petrosino S.
      • et al.
      Palmitoylethanolamide, a naturally occurring lipid, is an orally effective intestinal anti-inflammatory agent.
      PEA binds to peroxisome proliferator-activated receptor–α and inhibits the expression of S100B and Toll-like receptor 4 on enteric glia, to reduce inflammation induced by nuclear factor-κB.
      • Esposito G.
      • Capoccia E.
      • Turco F.
      • et al.
      Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-alpha activation.
      This is a complex system, because PEA, like anandamide, affects enteric glia and neurons in the gut, via different receptors on each cell type. PEA is not only degraded by FAAH but also N-acylethanolamine–hydrolyzing acid amidase.
      • Alhouayek M.
      • Bottemanne P.
      • Subramanian K.V.
      • et al.
      N-Acylethanolamine-hydrolyzing acid amidase inhibition increases colon N-palmitoylethanolamine levels and counteracts murine colitis.
      An N-acylethanolamine–hydrolyzing acid amidase inhibitor was shown to markedly reduce colitis, providing yet another therapeutic target for study.
      • Alhouayek M.
      • Bottemanne P.
      • Subramanian K.V.
      • et al.
      N-Acylethanolamine-hydrolyzing acid amidase inhibition increases colon N-palmitoylethanolamine levels and counteracts murine colitis.
      Although our discussion has focused on the GI tract, administration of the cannabinoid agonist CB13 to the CNS was reported to block development of colitis.
      • Fichna J.
      • Bawa M.
      • Thakur G.A.
      • et al.
      Cannabinoids alleviate experimentally induced intestinal inflammation by acting at central and peripheral receptors.
      The mechanism of this process has not been elucidated, but this remarkable finding indicates the importance of central and peripheral CB receptors in modulating gut functions and inflammation.

      Clinical Application of Endocannabinoid System Manipulation

      The significant role of the ECS in the GI tract makes it an appealing therapeutic target. However, the widespread distribution of the ECS and its extensive activities throughout the body
      • Maccarrone M.
      • Bab I.
      • Biro T.
      • et al.
      Endocannabinoid signaling at the periphery: 50 years after THC.
      make it a challenge to selectively alter in the gut without affecting other organ systems. Agonists of CB1 and CB2 (such as dronabinol and nabilone) are effective in treatment of nausea and vomiting and stimulating appetite, but are limited by their central side effects. Clinical studies using other CB receptor ligands have also proved disappointing. CB1 antagonists were thought to be promising for the treatment of obesity and metabolic syndrome,
      • Kipnes M.S.
      • Hollander P.
      • Fujioka K.
      • et al.
      A one-year study to assess the safety and efficacy of the CB1R inverse agonist taranabant in overweight and obese patients with type 2 diabetes.
      • Topol E.J.
      • Bousser M.G.
      • Fox K.A.
      • et al.
      Rimonabant for prevention of cardiovascular events (CRESCENDO): a randomised, multicentre, placebo-controlled trial.
      but were withdrawn from the market after some patients developed serious psychiatric side effects. Peripherally restricted agonist of CB1 and CB2 were not found to be effective in patients with pain, and produced CNS and cardiovascular side effects.
      • Kalliomaki J.
      • Annas P.
      • Huizar K.
      • et al.
      Evaluation of the analgesic efficacy and psychoactive effects of AZD1940, a novel peripherally acting cannabinoid agonist, in human capsaicin-induced pain and hyperalgesia.
      A selective agonist of CB2 was also examined for its analgesic potential and found to be rather ineffective, though well tolerated.
      • Ostenfeld T.
      • Price J.
      • Albanese M.
      • et al.
      A randomized, controlled study to investigate the analgesic efficacy of single doses of the cannabinoid receptor-2 agonist GW842166, ibuprofen or placebo in patients with acute pain following third molar tooth extraction.
      FAAH inhibitors appeared to be promising in preclinical studies, but human studies did not demonstrate efficacy, although these agents increased levels of anandamide in the target tissues.
      • Huggins J.P.
      • Smart T.S.
      • Langman S.
      • et al.
      An efficient randomised, placebo-controlled clinical trial with the irreversible fatty acid amide hydrolase-1 inhibitor PF-04457845, which modulates endocannabinoids but fails to induce effective analgesia in patients with pain due to osteoarthritis of the knee.
      Findings from preclinical studies have indicated that inhibitors of FAAH can promote cardiovascular injury, because of the role of the ECS in the heart,
      • Mukhopadhyay P.
      • Horvath B.
      • Rajesh M.
      • et al.
      Fatty acid amide hydrolase is a key regulator of endocannabinoid-induced myocardial tissue injury.
      • Hoyer F.F.
      • Khoury M.
      • Slomka H.
      • et al.
      Inhibition of endocannabinoid-degrading enzyme fatty acid amide hydrolase increases atherosclerotic plaque vulnerability in mice.
      • Lenglet S.
      • Thomas A.
      • Soehnlein O.
      • et al.
      Fatty acid amide hydrolase deficiency enhances intraplaque neutrophil recruitment in atherosclerotic mice.
      raising significant concerns about their chronic use, even if they are found to be effective. Most of these adverse effects are due to activation of CB1, as CB2 seems to protect the heart.
      • O'Sullivan S.E.
      Endocannabinoids and the cardiovascular system in health and disease.
      These studies paint a somewhat bleak picture for translation of promising preclinical findings to the clinic. Because the ECS is of such widespread importance, any systemic alterations involving the ECS will almost certainly produce challenges related to the drug target. Findings from clinical studies remind us that it is important to increase our understanding of the role of the ECS in maintenance of health and development of disease, and to better appreciate how this system interacts with other key regulatory systems.
      However, based on what is known about the role of the ECS in the control of the GI tract in health and disease, there are a number of important directions to consider in the development of therapeutic strategies to modulate the ECS. First, peripherally restricted CB1 antagonists are likely to increase gut motility and possibly reduce intestinal permeability—features that could make them useful therapeutics, especially given their beneficial effects in patients with diabetes.
      • Pacher P.
      • Kunos G.
      Modulating the endocannabinoid system in human health and disease—successes and failures.
      Agonists of CB2 could reduce inflammation in the GI tract and limit abnormally accelerated motility. Another approach could be to use cannabinoid ligands that are not absorbed and act only on the epithelium.
      • Rousseaux C.
      • Thuru X.
      • Gelot A.
      • et al.
      Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors.

      Future Directions

      The ECS is an important regulator of intestinal function and the brain–gut axis. It is a widely distributed homeostatic system that generally inhibits neural activity in pathways involved in the physiologic regulation of the GI tract. Important gut functions, including visceral sensation, pain, motility, and inflammation, are regulated by the ECS. However, there are many areas where we lack a detailed understanding of the distribution and function of the various components of the ECS, notably the biosynthetic enzymes for anandamide and 2-AG.
      Genetic and/or epigenetic alterations that affect activities of the ECS could be involved in the pathogenesis of IBS. As we have outlined, alterations of the ECS can provide a link between stress and visceral pain, a well-recognized feature of many GI conditions. Inflammation of the gut alters the expression of degradative enzymes of the ECS, leading to marked changes in the local levels of these pleiotropic lipid mediators. Far more work is required to better understand the involvement of the ECS in GI diseases.
      The glucocorticoid receptor regulates expression of CNR1, and chronic stress is associated with increased DMNT1-mediated methylation at the promoters of genes that encode the glucocorticoid receptor (NR3C1) and CB1 (CNR1), resulting in decreased expression of the glucocorticoid receptor and CB1. Chronic stress-induced changes in this pathway are region- and cell-specific and might be targeted by therapeutic agents. Chronic stress and chronic cannabis use affect ECS regulation of the circadian HPA pathway, so chronic stress can affect potential circadian and ultradian transcriptional regulation of CNR1 in a region- and cell-specific manner. Cortisol (human) and corticosterone (rodent) have been shown to promote circadian and ultradian bursts of transcriptional activity in many genes, including the CLOCK gene family; this rhythm is disrupted in patients with stress-related mental health, GI, or immune disorders.
      • Wiley J.W.
      • Higgins G.A.
      • Athey B.D.
      Stress and glucocorticoid receptor transcriptional programming in time and space: implications for the brain-gut axis.
      Epigenetic factors determine how the glucocorticoid receptor interacts with target genes, including CNR1. Dysregulation of the central and peripheral glucocorticoid receptor regulome has potentially significant consequences for stress-related disorders that affect the brain–gut axis and ECS.
      Cannabinoids are prescribed for GI conditions, particularly nausea and vomiting, as an appetite stimulant and for the treatment of visceral pain. There is evidence that direct or indirect activation of CB1, and possibly CB2, inhibits visceral sensitivity and pain in rodents. Central side effects, such as drowsiness and heightened awareness, have been reported. Recently developed ligands of CB2 and peripherally acting ligands of CB1 and CB2 have the potential to alleviate GI symptoms without CNS side effects. Future research will focus on the development of compounds that either act directly at the CB receptors or alter ligand availability in a region-specific manner to minimize side effects on brain function. FAAH, MAGL, and N-acylethanolamine–hydrolyzing acid amidase appear to be promising therapeutic targets based on findings from animal studies, but their widespread physiologic functions could limit our ability to alter their activity for treatment of chronic GI conditions.
      With the increasing use of cannabis in Western society, there is a need to better understand the effects of the many components of this complex plant on gut functions in health and disease. A full understanding of the ECS will advance our knowledge of the brain–gut axis and could lead to new avenues for the treatment of many GI disorders.

      Acknowledgments

      Keith A. Sharkey is the Crohn’s Colitis Canada Chair in Inflammatory Bowel Disease Research at the University of Calgary.
      Keith A. Sharkey and John W. Wiley contributed equally to the research, writing, illustration, and revision of the manuscript.

      References

        • Grinspoon L.
        • Bakalar J.B.
        Marihuana, the Forbidden Medicine.
        Yale University Press, New Haven1997
        • Aviello G.
        • Romano B.
        • Izzo A.A.
        Cannabinoids and gastrointestinal motility: animal and human studies.
        Eur Rev Med Pharmacol Sci. 2008; 12: 81-93
        • Izzo A.A.
        • Sharkey K.A.
        Cannabinoids and the gut: new developments and emerging concepts.
        Pharmacol Ther. 2010; 126: 21-38
        • Sharkey K.A.
        • Darmani N.A.
        • Parker L.A.
        Regulation of nausea and vomiting by cannabinoids and the endocannabinoid system.
        Eur J Pharmacol. 2014; 722: 134-146
        • Abalo R.
        • Vera G.
        • Lopez-Perez A.E.
        • et al.
        The gastrointestinal pharmacology of cannabinoids: focus on motility.
        Pharmacology. 2012; 90: 1-10
        • Alhouayek M.
        • Muccioli G.G.
        The endocannabinoid system in inflammatory bowel diseases: from pathophysiology to therapeutic opportunity.
        Trends Mol Med. 2012; 18: 615-625
        • Whiting P.F.
        • Wolff R.F.
        • Deshpande S.
        • et al.
        Cannabinoids for medical use: a systematic review and meta-analysis.
        JAMA. 2015; 313: 2456-2473
        • Maccarrone M.
        • Bab I.
        • Biro T.
        • et al.
        Endocannabinoid signaling at the periphery: 50 years after THC.
        Trends Pharmacol Sci. 2015; 36: 277-296
        • Di Marzo V.
        • De Petrocellis L.
        Why do cannabinoid receptors have more than one endogenous ligand?.
        Philos Trans R Soc Lond B Biol Sci. 2012; 367: 3216-3228
        • Cani P.D.
        Crosstalk between the gut microbiota and the endocannabinoid system: impact on the gut barrier function and the adipose tissue.
        Clin Microbiol Infect. 2012; 18: 50-53
        • Hill M.N.
        • Patel S.
        • Campolongo P.
        • et al.
        Functional interactions between stress and the endocannabinoid system: from synaptic signaling to behavioral output.
        J Neurosci. 2010; 30: 14980-14986
        • Hill M.N.
        • Tasker J.G.
        Endocannabinoid signaling, glucocorticoid-mediated negative feedback, and regulation of the hypothalamic-pituitary-adrenal axis.
        Neuroscience. 2012; 204: 5-16
        • Russo E.B.
        Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects.
        Br J Pharmacol. 2011; 163: 1344-1364
        • Gertsch J.
        • Pertwee R.G.
        • Di Marzo V.
        Phytocannabinoids beyond the Cannabis plant—do they exist?.
        Br J Pharmacol. 2010; 160: 523-529
        • De Petrocellis L.
        • Orlando P.
        • Moriello A.S.
        • et al.
        Cannabinoid actions at TRPV channels: effects on TRPV3 and TRPV4 and their potential relevance to gastrointestinal inflammation.
        Acta Physiol (Oxf). 2012; 204: 255-266
        • Izzo A.A.
        • Capasso R.
        • Aviello G.
        • et al.
        Inhibitory effect of cannabichromene, a major non-psychotropic cannabinoid extracted from Cannabis sativa, on inflammation-induced hypermotility in mice.
        Br J Pharmacol. 2012; 166: 1444-1460
        • Hill A.J.
        • Williams C.M.
        • Whalley B.J.
        • et al.
        Phytocannabinoids as novel therapeutic agents in CNS disorders.
        Pharmacol Ther. 2012; 133: 79-97
        • Di Marzo V.
        • Piscitelli F.
        The endocannabinoid system and its modulation by phytocannabinoids.
        Neurotherapeutics. 2015; 12: 692-698
        • Katona I.
        • Freund T.F.
        Multiple functions of endocannabinoid signaling in the brain.
        Annu Rev Neurosci. 2012; 35: 529-558
        • Di Marzo V.
        • Fontana A.
        • Cadas H.
        • et al.
        Formation and inactivation of endogenous cannabinoid anandamide in central neurons.
        Nature. 1994; 372: 686-691
        • Blankman J.L.
        • Cravatt B.F.
        Chemical probes of endocannabinoid metabolism.
        Pharmacol Rev. 2013; 65: 849-871
        • Deutsch D.G.
        • Ueda N.
        • Yamamoto S.
        The fatty acid amide hydrolase (FAAH).
        Prostaglandins Leukot Essent Fatty Acids. 2002; 66: 201-210
        • Marquez L.
        • Suarez J.
        • Iglesias M.
        • et al.
        Ulcerative colitis induces changes on the expression of the endocannabinoid system in the human colonic tissue.
        PLoS One. 2009; 4: e6893
        • Blankman J.L.
        • Simon G.M.
        • Cravatt B.F.
        A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.
        Chem Biol. 2007; 14: 1347-1356
        • Hermanson D.J.
        • Gamble-George J.C.
        • Marnett L.J.
        • et al.
        Substrate-selective COX-2 inhibition as a novel strategy for therapeutic endocannabinoid augmentation.
        Trends Pharmacol Sci. 2014; 35: 358-367
        • Alhouayek M.
        • Muccioli G.G.
        COX-2-derived endocannabinoid metabolites as novel inflammatory mediators.
        Trends Pharmacol Sci. 2014; 35: 284-292
        • Trautmann S.M.
        • Sharkey K.A.
        The endocannabinoid system and its role in regulating the intrinsic neural circuitry of the gastrointestinal tract.
        Int Rev Neurobiol. 2015; 125: 85-126
        • Starowicz K.
        • Nigam S.
        • Di Marzo V.
        Biochemistry and pharmacology of endovanilloids.
        Pharmacol Ther. 2007; 114: 13-33
        • Wright K.
        • Rooney N.
        • Feeney M.
        • et al.
        Differential expression of cannabinoid receptors in the human colon: cannabinoids promote epithelial wound healing.
        Gastroenterology. 2005; 129: 437-453
        • Wright K.L.
        • Duncan M.
        • Sharkey K.A.
        Cannabinoid CB2 receptors in the gastrointestinal tract: a regulatory system in states of inflammation.
        Br J Pharmacol. 2008; 153: 263-270
        • Rousseaux C.
        • Thuru X.
        • Gelot A.
        • et al.
        Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors.
        Nat Med. 2007; 13: 35-37
        • Aguilera M.
        • Vergara P.
        • Martinez V.
        Stress and antibiotics alter luminal and wall-adhered microbiota and enhance the local expression of visceral sensory-related systems in mice.
        Neurogastroenterol Motil. 2013; 25: e515-e529
        • Ringel-Kulka T.
        • Goldsmith J.R.
        • Carroll I.M.
        • et al.
        Lactobacillus acidophilus NCFM affects colonic mucosal opioid receptor expression in patients with functional abdominal pain—a randomised clinical study.
        Aliment Pharmacol Ther. 2014; 40: 200-207
        • Storr M.A.
        • Sharkey K.A.
        The endocannabinoid system and gut-brain signalling.
        Curr Opin Pharmacol. 2007; 7: 575-582
        • Hohmann A.G.
        • Herkenham M.
        Localization of central cannabinoid CB1 receptor messenger RNA in neuronal subpopulations of rat dorsal root ganglia: a double-label in situ hybridization study.
        Neuroscience. 1999; 90: 923-931
        • Bridges D.
        • Rice A.S.
        • Egertova M.
        • et al.
        Localisation of cannabinoid receptor 1 in rat dorsal root ganglion using in situ hybridisation and immunohistochemistry.
        Neuroscience. 2003; 119: 803-812
        • Ahluwalia J.
        • Urban L.
        • Capogna M.
        • et al.
        Cannabinoid 1 receptors are expressed in nociceptive primary sensory neurons.
        Neuroscience. 2000; 100: 685-688
        • Amaya F.
        • Shimosato G.
        • Kawasaki Y.
        • et al.
        Induction of CB1 cannabinoid receptor by inflammation in primary afferent neurons facilitates antihyperalgesic effect of peripheral CB1 agonist.
        Pain. 2006; 124: 175-183
        • Malik Z.
        • Baik D.
        • Schey R.
        The role of cannabinoids in regulation of nausea and vomiting, and visceral pain.
        Curr Gastroenterol Rep. 2015; 17: 429
        • Turcotte D.
        • Le Dorze J.A.
        • Esfahani F.
        • et al.
        Examining the roles of cannabinoids in pain and other therapeutic indications: a review.
        Expert Opin Pharmacother. 2010; 11: 17-31
        • Hong S.
        • Fan J.
        • Kemmerer E.S.
        • et al.
        Reciprocal changes in vanilloid (TRPV1) and endocannabinoid (CB1) receptors contribute to visceral hyperalgesia in the water avoidance stressed rat.
        Gut. 2009; 58: 202-210
        • Hong S.
        • Zheng G.
        • Wu X.
        • et al.
        Corticosterone mediates reciprocal changes in CB 1 and TRPV1 receptors in primary sensory neurons in the chronically stressed rat.
        Gastroenterology. 2011; 140: 627-637 e4
        • Bestor T.H.
        The DNA methyltransferases of mammals.
        Hum Mol Genet. 2000; 9: 2395-2402
        • Miranda T.B.
        • Jones P.A.
        DNA methylation: the nuts and bolts of repression.
        J Cell Physiol. 2007; 213: 384-390
        • Karlic R.
        • Chung H.R.
        • Lasserre J.
        • et al.
        Histone modification levels are predictive for gene expression.
        Proc Natl Acad Sci U S A. 2010; 107: 2926-2931
        • Hong S.
        • Zheng G.
        • Wiley J.W.
        Epigenetic regulation of genes that modulate chronic stress-induced visceral pain in the peripheral nervous system.
        Gastroenterology. 2015; 148: 148-157 e7
        • Morena M.
        • Patel S.
        • Bains J.S.
        • et al.
        Neurobiological Interactions between stress and the endocannabinoid system.
        Neuropsychopharmacology. 2016; 41: 80-102
        • Lomazzo E.
        • Bindila L.
        • Remmers F.
        • et al.
        Therapeutic potential of inhibitors of endocannabinoid degradation for the treatment of stress-related hyperalgesia in an animal model of chronic pain.
        Neuropsychopharmacology. 2015; 40: 488-501
        • Lau B.K.
        • Drew G.M.
        • Mitchell V.A.
        • et al.
        Endocannabinoid modulation by FAAH and monoacylglycerol lipase within the analgesic circuitry of the periaqueductal grey.
        Br J Pharmacol. 2014; 171: 5225-5236
        • Wang Z.
        • Bradesi S.
        • Maarek J.M.
        • et al.
        Regional brain activation in conscious, nonrestrained rats in response to noxious visceral stimulation.
        Pain. 2008; 138: 233-243
        • Adamson Barnes N.S.
        • Mitchell V.A.
        • Kazantzis N.P.
        • et al.
        Actions of the dual FAAH/MAGL inhibitor JZL195 in a murine neuropathic pain model.
        Br J Pharmacol. 2016; 173: 77-87
        • Sakin Y.S.
        • Dogrul A.
        • Ilkaya F.
        • et al.
        The effect of FAAH, MAGL, and Dual FAAH/MAGL inhibition on inflammatory and colorectal distension-induced visceral pain models in Rodents.
        Neurogastroenterol Motil. 2015; 27: 936-944
        • Marco E.M.
        • Echeverry-Alzate V.
        • Lopez-Moreno J.A.
        • et al.
        Consequences of early life stress on the expression of endocannabinoid-related genes in the rat brain.
        Behav Pharmacol. 2014; 25: 547-556
        • Moloney R.D.
        • Stilling R.M.
        • Dinan T.G.
        • et al.
        Early-life stress-induced visceral hypersensitivity and anxiety behavior is reversed by histone deacetylase inhibition.
        Neurogastroenterol Motil. 2015; 27: 1831-1836
        • Gschossmann J.M.
        • Buenger L.
        • Adam B.
        • et al.
        Diurnal variation of abdominal motor responses to colorectal distension and plasma cortisol levels in rats.
        Neurogastroenterol Motil. 2001; 13: 585-589
        • Chang L.
        • Sundaresh S.
        • Elliott J.
        • et al.
        Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in irritable bowel syndrome.
        Neurogastroenterol Motil. 2009; 21: 149-159
        • Liedhegner E.S.
        • Sasman A.
        • Hillard C.J.
        Brain region-specific changes in N-acylethanolamine contents with time of day.
        J Neurochem. 2014; 128: 491-506
        • Patel S.
        • Roelke C.T.
        • Rademacher D.J.
        • et al.
        Endocannabinoid signaling negatively modulates stress-induced activation of the hypothalamic-pituitary-adrenal axis.
        Endocrinology. 2004; 145: 5431-5438
        • Saber-Tehrani A.
        • Naderi N.
        • Hosseini Najarkolaei A.
        • et al.
        Cannabinoids and their interactions with diazepam on modulation of serum corticosterone concentration in male mice.
        Neurochem Res. 2010; 35: 60-66
        • Cone E.J.
        • Johnson R.E.
        • Moore J.D.
        • et al.
        Acute effects of smoking marijuana on hormones, subjective effects and performance in male human subjects.
        Pharmacol Biochem Behav. 1986; 24: 1749-1754
        • Kleinloog D.
        • Liem-Moolenaar M.
        • Jacobs G.
        • et al.
        Does olanzapine inhibit the psychomimetic effects of Delta(9)-tetrahydrocannabinol?.
        J Psychopharmacol. 2012; 26: 1307-1316
        • Klumpers L.E.
        • Beumer T.L.
        • van Hasselt J.G.
        • et al.
        Novel Delta(9) -tetrahydrocannabinol formulation Namisol(R) has beneficial pharmacokinetics and promising pharmacodynamic effects.
        Br J Clin Pharmacol. 2012; 74: 42-53
        • D'Souza D.C.
        • Ranganathan M.
        • Braley G.
        • et al.
        Blunted psychotomimetic and amnestic effects of delta-9-tetrahydrocannabinol in frequent users of cannabis.
        Neuropsychopharmacology. 2008; 33: 2505-2516
        • Ranganathan M.
        • Braley G.
        • Pittman B.
        • et al.
        The effects of cannabinoids on serum cortisol and prolactin in humans.
        Psychopharmacology (Berl). 2009; 203: 737-744
        • King G.R.
        • Ernst T.
        • Deng W.
        • et al.
        Altered brain activation during visuomotor integration in chronic active cannabis users: relationship to cortisol levels.
        J Neurosci. 2011; 31: 17923-17931
        • Somaini L.
        • Manfredini M.
        • Amore M.
        • et al.
        Psychobiological responses to unpleasant emotions in cannabis users.
        Eur Arch Psychiatry Clin Neurosci. 2012; 262: 47-57
        • van Leeuwen A.P.
        • Creemers H.E.
        • Greaves-Lord K.
        • et al.
        Hypothalamic-pituitary-adrenal axis reactivity to social stress and adolescent cannabis use: the TRAILS study.
        Addiction. 2011; 106: 1484-1492
        • Van Sickle M.D.
        • Duncan M.
        • Kingsley P.J.
        • et al.
        Identification and functional characterization of brainstem cannabinoid CB2 receptors.
        Science. 2005; 310: 329-332
        • Napadow V.
        • Sheehan J.D.
        • Kim J.
        • et al.
        The brain circuitry underlying the temporal evolution of nausea in humans.
        Cereb Cortex. 2013; 23: 806-813
        • Sclocco R.
        • Kim J.
        • Garcia R.G.
        • et al.
        Brain circuitry supporting multi-organ autonomic outflow in response to nausea.
        Cereb Cortex. 2016; 26: 485-497
        • Sticht M.A.
        • Limebeer C.L.
        • Rafla B.R.
        • et al.
        Endocannabinoid regulation of nausea is mediated by 2-arachidonoylglycerol (2-AG) in the rat visceral insular cortex.
        Neuropharmacology. 2016; 102: 92-102
        • Parker L.A.
        • Rock E.M.
        • Sticht M.A.
        • et al.
        Cannabinoids suppress acute and anticipatory nausea in preclinical rat models of conditioned gaping.
        Clin Pharmacol Ther. 2015; 97: 559-561
        • Parker L.A.
        Conditioned flavor avoidance and conditioned gaping: rat models of conditioned nausea.
        Eur J Pharmacol. 2014; 722: 122-133
        • Sticht M.A.
        • Limebeer C.L.
        • Rafla B.R.
        • et al.
        Intra-visceral insular cortex 2-arachidonoylglycerol, but not N-arachidonoylethanolamide, suppresses acute nausea-induced conditioned gaping in rats.
        Neuroscience. 2015; 286: 338-344
        • Allen J.H.
        • de Moore G.M.
        • Heddle R.
        • et al.
        Cannabinoid hyperemesis: cyclical hyperemesis in association with chronic cannabis abuse.
        Gut. 2004; 53: 1566-1570
        • Lee L.Y.
        • Abbott L.
        • Moodie S.
        • et al.
        Cyclic vomiting syndrome in 28 patients: demographics, features and outcomes.
        Eur J Gastroenterol Hepatol. 2012; 24: 939-943
        • Chang Y.H.
        • Windish D.M.
        Cannabinoid hyperemesis relieved by compulsive bathing.
        Mayo Clin Proc. 2009; 84: 76-78
        • Soriano-Co M.
        • Batke M.
        • Cappell M.S.
        The cannabis hyperemesis syndrome characterized by persistent nausea and vomiting, abdominal pain, and compulsive bathing associated with chronic marijuana use: a report of eight cases in the United States.
        Dig Dis Sci. 2010; 55: 3113-3119
        • Wallace E.A.
        • Andrews S.E.
        • Garmany C.L.
        • et al.
        Cannabinoid hyperemesis syndrome: literature review and proposed diagnosis and treatment algorithm.
        South Med J. 2011; 104: 659-664
        • Darmani N.A.
        Cannabinoid-induced hyperemesis: a conundrum—from clinical recognition to basic science mechanisms.
        Pharmaceuticals. 2010; 3: 2163-2177
        • Bolognesi M.
        • Di Pascoli M.
        • Verardo A.
        • et al.
        Splanchnic vasodilation and hyperdynamic circulatory syndrome in cirrhosis.
        World J Gastroenterol. 2014; 20: 2555-2563
        • Camilleri M.
        • Kolar G.J.
        • Vazquez-Roque M.I.
        • et al.
        Cannabinoid receptor 1 gene and irritable bowel syndrome: phenotype and quantitative traits.
        Am J Physiol Gastrointest Liver Physiol. 2013; 304: G553-G560
        • Park J.M.
        • Choi M.G.
        • Cho Y.K.
        • et al.
        Cannabinoid receptor 1 gene polymorphism and irritable bowel syndrome in the Korean population: a hypothesis-generating study.
        J Clin Gastroenterol. 2011; 45: 45-49
        • Camilleri M.
        • Carlson P.
        • McKinzie S.
        • et al.
        Genetic variation in endocannabinoid metabolism, gastrointestinal motility, and sensation.
        Am J Physiol Gastrointest Liver Physiol. 2008; 294: G13-G19
        • Fichna J.
        • Wood J.T.
        • Papanastasiou M.
        • et al.
        Endocannabinoid and cannabinoid-like fatty acid amide levels correlate with pain-related symptoms in patients with IBS-D and IBS-C: a pilot study.
        PLoS One. 2013; 8: e85073
        • Vianna C.R.
        • Donato Jr., J.
        • Rossi J.
        • et al.
        Cannabinoid receptor 1 in the vagus nerve is dispensable for body weight homeostasis but required for normal gastrointestinal motility.
        J Neurosci. 2012; 32: 10331-10337
        • Keenan C.M.
        • Storr M.A.
        • Thakur G.A.
        • et al.
        AM841, a covalent cannabinoid ligand, powerfully slows gastrointestinal motility in normal and stressed mice in a peripherally restricted manner.
        Br J Pharmacol. 2015; 172: 2406-2418
        • Abalo R.
        • Chen C.
        • Vera G.
        • et al.
        In vitro and non-invasive in vivo effects of the cannabinoid-1 receptor agonist AM841 on gastrointestinal motor function in the rat.
        Neurogastroenterol Motil. 2015; 27: 1721-1735
        • Cluny N.L.
        • Keenan C.M.
        • Duncan M.
        • et al.
        Naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (SAB378), a peripherally restricted cannabinoid CB1/CB2 receptor agonist, inhibits gastrointestinal motility but has no effect on experimental colitis in mice.
        J Pharmacol Exp Ther. 2010; 334: 973-980
        • Bashashati M.
        • Nasser Y.
        • Keenan C.M.
        • et al.
        Inhibiting endocannabinoid biosynthesis: a novel approach to the treatment of constipation.
        Br J Pharmacol. 2015; 172: 3099-3111
        • Fichna J.
        • Salaga M.
        • Stuart J.
        • et al.
        Selective inhibition of FAAH produces antidiarrheal and antinociceptive effect mediated by endocannabinoids and cannabinoid-like fatty acid amides.
        Neurogastroenterol Motil. 2014; 26: 470-481
        • Bashashati M.
        • Storr M.A.
        • Nikas S.P.
        • et al.
        Inhibiting fatty acid amide hydrolase normalizes endotoxin-induced enhanced gastrointestinal motility in mice.
        Br J Pharmacol. 2012; 165: 1556-1571
        • Mukhopadhyay P.
        • Horvath B.
        • Rajesh M.
        • et al.
        Fatty acid amide hydrolase is a key regulator of endocannabinoid-induced myocardial tissue injury.
        Free Radic Biol Med. 2011; 50: 179-195
        • Hoyer F.F.
        • Khoury M.
        • Slomka H.
        • et al.
        Inhibition of endocannabinoid-degrading enzyme fatty acid amide hydrolase increases atherosclerotic plaque vulnerability in mice.
        J Mol Cell Cardiol. 2014; 66: 126-132
        • Lenglet S.
        • Thomas A.
        • Soehnlein O.
        • et al.
        Fatty acid amide hydrolase deficiency enhances intraplaque neutrophil recruitment in atherosclerotic mice.
        Arterioscler Thromb Vasc Biol. 2013; 33: 215-223
        • Massa F.
        • Marsicano G.
        • Hermann H.
        • et al.
        The endogenous cannabinoid system protects against colonic inflammation.
        J Clin Invest. 2004; 113: 1202-1209
        • Storr M.A.
        • Keenan C.M.
        • Emmerdinger D.
        • et al.
        Targeting endocannabinoid degradation protects against experimental colitis in mice: involvement of CB1 and CB2 receptors.
        J Mol Med (Berl). 2008; 86: 925-936
        • Salaga M.
        • Mokrowiecka A.
        • Zakrzewski P.K.
        • et al.
        Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition of fatty acid amide hydrolase (FAAH).
        J Crohns Colitis. 2014; 8: 998-1009
        • Alhouayek M.
        • Lambert D.M.
        • Delzenne N.M.
        • et al.
        Increasing endogenous 2-arachidonoylglycerol levels counteracts colitis and related systemic inflammation.
        FASEB J. 2011; 25: 2711-2721
        • Storr M.
        • Emmerdinger D.
        • Diegelmann J.
        • et al.
        The role of fatty acid hydrolase gene variants in inflammatory bowel disease.
        Aliment Pharmacol Ther. 2009; 29: 542-551
        • Suarez J.
        • Romero-Zerbo Y.
        • Marquez L.
        • et al.
        Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids.
        PLoS One. 2012; 7: e37729
        • D'Argenio G.
        • Valenti M.
        • Scaglione G.
        • et al.
        Up-regulation of anandamide levels as an endogenous mechanism and a pharmacological strategy to limit colon inflammation.
        FASEB J. 2006; 20: 568-570
        • Di Sabatino A.
        • Battista N.
        • Biancheri P.
        • et al.
        The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease.
        Mucosal Immunol. 2011; 4: 574-583
        • Sasso O.
        • Migliore M.
        • Habrant D.
        • et al.
        Multitarget fatty acid amide hydrolase/cyclooxygenase blockade suppresses intestinal inflammation and protects against nonsteroidal anti-inflammatory drug-dependent gastrointestinal damage.
        FASEB J. 2015; 29: 2616-2627
        • Darmani N.A.
        • Izzo A.A.
        • Degenhardt B.
        • et al.
        Involvement of the cannabimimetic compound, N-palmitoyl-ethanolamine, in inflammatory and neuropathic conditions: review of the available pre-clinical data, and first human studies.
        Neuropharmacology. 2005; 48: 1154-1163
        • Borrelli F.
        • Romano B.
        • Petrosino S.
        • et al.
        Palmitoylethanolamide, a naturally occurring lipid, is an orally effective intestinal anti-inflammatory agent.
        Br J Pharmacol. 2015; 172: 142-158
        • Esposito G.
        • Capoccia E.
        • Turco F.
        • et al.
        Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-alpha activation.
        Gut. 2014; 63: 1300-1312
        • Alhouayek M.
        • Bottemanne P.
        • Subramanian K.V.
        • et al.
        N-Acylethanolamine-hydrolyzing acid amidase inhibition increases colon N-palmitoylethanolamine levels and counteracts murine colitis.
        FASEB J. 2015; 29: 650-661
        • Fichna J.
        • Bawa M.
        • Thakur G.A.
        • et al.
        Cannabinoids alleviate experimentally induced intestinal inflammation by acting at central and peripheral receptors.
        PLoS One. 2014; 9: e109115
        • Kipnes M.S.
        • Hollander P.
        • Fujioka K.
        • et al.
        A one-year study to assess the safety and efficacy of the CB1R inverse agonist taranabant in overweight and obese patients with type 2 diabetes.
        Diabetes Obes Metab. 2010; 12: 517-531
        • Topol E.J.
        • Bousser M.G.
        • Fox K.A.
        • et al.
        Rimonabant for prevention of cardiovascular events (CRESCENDO): a randomised, multicentre, placebo-controlled trial.
        Lancet. 2010; 376: 517-523
        • Kalliomaki J.
        • Annas P.
        • Huizar K.
        • et al.
        Evaluation of the analgesic efficacy and psychoactive effects of AZD1940, a novel peripherally acting cannabinoid agonist, in human capsaicin-induced pain and hyperalgesia.
        Clin Exp Pharmacol Physiol. 2013; 40: 212-218
        • Ostenfeld T.
        • Price J.
        • Albanese M.
        • et al.
        A randomized, controlled study to investigate the analgesic efficacy of single doses of the cannabinoid receptor-2 agonist GW842166, ibuprofen or placebo in patients with acute pain following third molar tooth extraction.
        Clin J Pain. 2011; 27: 668-676
        • Huggins J.P.
        • Smart T.S.
        • Langman S.
        • et al.
        An efficient randomised, placebo-controlled clinical trial with the irreversible fatty acid amide hydrolase-1 inhibitor PF-04457845, which modulates endocannabinoids but fails to induce effective analgesia in patients with pain due to osteoarthritis of the knee.
        Pain. 2012; 153: 1837-1846
        • O'Sullivan S.E.
        Endocannabinoids and the cardiovascular system in health and disease.
        Handb Exp Pharmacol. 2015; 231: 393-422
        • Pacher P.
        • Kunos G.
        Modulating the endocannabinoid system in human health and disease—successes and failures.
        FEBS J. 2013; 280: 1918-1943
        • Wiley J.W.
        • Higgins G.A.
        • Athey B.D.
        Stress and glucocorticoid receptor transcriptional programming in time and space: implications for the brain-gut axis.
        Neurogastroenterol Motil. 2016; 28: 12-25
        • Di Marzo V.
        • Stella N.
        • Zimmer A.
        Endocannabinoid signalling and the deteriorating brain.
        Nat Rev Neurosci. 2015; 16: 30-42
        • Krugers H.J.
        • Hoogenraad C.C.
        • Groc L.
        Stress hormones and AMPA receptor trafficking in synaptic plasticity and memory.
        Nat Rev Neurosci. 2010; 11: 675-681
        • Sticht M.A.
        • Rock E.M.
        • Limebeer C.L.
        • et al.
        Endocannabinoid mechanisms influencing nausea.
        Int Rev Neurobiol. 2015; 125: 127-162
        • Lomax A.E.
        • Linden D.R.
        • Mawe G.M.
        • et al.
        Effects of gastrointestinal inflammation on enteroendocrine cells and enteric neural reflex circuits.
        Auton Neurosci. 2006; 126–127: 250-257